| With the developments in technology and the increase of the global population,the demand of plastics is greatly increasing.Because of some characteristics of polystyrene(PS),e.g.the low cost,low density,and great translucency,it has been widely used in human life,industries and other aspects.However,the stability of polystyrene makes it very hard to degrade in the environment.Polystyrene waste has accumulated in the natural environment and caused serious pollution.Nowadays,some researchers found that the superworms(larvae of Zophobas morio)and mealworms(larvae of Tenebrio molitor)could feed on plastics,and these reports might provide new ways for polystyrene biodegradation.This study aimed to find biodegrading strains from the guts of polystyrene-eating worms.After superworms were fed on polystyrene,the biodegrading strains were isolated from the guts of mealworms with polystyrene as a sole carbon source.Then visual observation and some chemical analysis experiments of degraded polystyrene were performed to test the biodegrading abilities of isolated strains;16S r RNA high through-put sequencing was used to study the differences in gut microbiota of mealworms in different diets;in addition,biodegrading strains in gut microbiota of mealworms were isolated from hyperhaline mineral medium which contains polystyrene as a sole carbon,then visual observation of degraded polystyrene and other chemical analysis methods were used to test the biodegrading abilities of isolated strains.The detailed achievements are described as below.1.Polystyrene-degrading bacterial strains were isolated from guts of superworms and characterized.Two bacterial strains(Citrobacter sp.PSDM1 and Klebsiella sp.PSDM2)were isolated from the gut microbial enrichment with PS as a sole carbon source.Viable biofilm formed by PSDM1 and PSDM2 was observed in 7 d,and after incubation with isolated strains28 d,a lot of pores and deterioration on polystyrene films were observed through scanning electron microscope(SEM)and atomic force microscope(AFM).The bacterial cell surface hydrophobicity test showed that PSDM1 and PSDM2 both have bacterial hydrophobicity,and PSDM2 has higher hydrophobicity of cell surface.Using Fourier transform infrared spectroscopy(FTIR),thermo gravimetric analysis(TGA)and ~1H nuclear magnetic resonance(~1H NMR)to analyze and compare untreated PS and degrading PS,the results demonstrated there were some new components appearing.The weight loss in 28 d showed PSDM1 could degrade 6.52%added PS and PSDM2 could degrade 7.33%added PS.All the results above indicated that the gut microbiota of superworms contains PS-degrading strains.2.The gut microbiota of mealworms fed on PS was analyzed and compared with that of mealworms fed on a normal diet.The mealworms were grouped into 3 experimental cohorts,i.e.bran-eating,styroform-eating and starvation groups,and were reared for different periods.After the rearing,guts were dissected from mealworms,and subjected to DNA extraction and PCR amplification of bacterial 16S r RNA genes.The amplicons were then analysed by high-through put sequencing to analyze the gut microbiota of mealworms.The results demonstrated that there were 1 713 OTUs in 33 samples,and most of the length of the sequences were in 420-440 bp.Halomonos was the most dominate genus in bran-eating and styroform-eating samples,and the gut microbiota in bran-eating and styroform-eating samples were extremely similar and both were very different from those of starved worms.Comamonadaceae and Enterobacteriaceae might play important roles in plastic-eating mealworms.3.PS-degrading bacterial strain was isolated from guts of mealworms in hyperhaline medium and confirmed its degrading ability.One bacteria strains Staphylococcus sp.HS.S was isolated from the gut microbial enrichment supplied PS as a sole carbon source.Viable biofilm and pores formed by HS.S was observed in 7 d.After incubated with HS.S in 28 d,a lot of damages and pits on PS surfaces were observed under SEM and AFM.FTIR,TGA and~1H NMR were used to compare the untreated and degraded PS,and the results showed comparing to the untreated PS,the chemical structure of degraded PS changed.The weight loss of PS after degraded by HS.S in 28 d was 7.15%.Number-average molecular weight and weight-average molecular weight of degraded PS both decreased largely,and the average molecular weight of degraded PS shifted to lower molecular weight.All of these results clearly demonstrated that mealworms and superworms harbor specific polystyrene-degrading bacteria in the guts,which plausibly contribute to the PS eating behavior of the worms. |
